Directed differentiation of human embryonic stem cells into keratinocyte progenitors in vitro: an attempt with promise of clinical use.

Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus, efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA), bone morphogenetic protein-4 (BMP4), and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA, AA, and BMP4; the third was commercial-defined keratinocyte serum-free medium, which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR, immunofluorescence, and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology, while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12, all P < 0.05) After hESC induction by AA+BMP4+RA, CK14 mRNA expression was upregulated after day 21, peaking by 35 d of differentiation. Combined RA, BMP4, and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering.